Clara cell degranulate in response to toxic doses of inhaled oxidant pollutants prior to entry into the cell cycle. Human and animals studies investigating the pulmonary response to chronic oxidant exposure demonstrate increases in activity of antioxidant enzymes and/or elevation of clara cell secretory protein (CCSP) content. Changes in CCSP content of chronic ozone-exposed rats are attributed to adaptive remodeling of airways involving hyperplasia of clara cells. The investigators hypothesize that oxidant-induced clara cell secretion protects the airway epithelium from injury. This hypothesis is supported by our studies characterizing the phenotype of CCSP deficient mice (CCSP [-/-]), which have ultrastructural changes to clara cell secretory apparatus, including loss of secretory granules, and exhibit increased sensitivity to injury by inhaled oxidant gases. The investigators propose to define mechanisms by which clara cell secretion participate in the acute physiologic response to ozone exposure.
Aim 1 addresses the hypothesis that ozone-induced clara cell secretion is an immediate protective response. Kinetics of ozone-induced clara cell secretion will be examined by measurement of degranulation in wild-type (WT) mice, and changes in lung and BAL pool sizes of secretory proteins CC26 and tryptase clara (trypC) in WT and CCSP [-/-] mice. Kinetics of secretion will be correlated with measures of lung injury including histopathology and expression of mRNA's for c-jun, TNF-alpha, MT and IL6 (injury-related genes).
Aim 2 will investigate the cellular basis for alterations in clara cell secretion in CCSP [-/-] mice. No changes were observed in steady-state levels of mRNA's normally expressed in clara cell (those coding for CYP2F2, SP-A and SP-B). Therefore, they hypothesize that clara cells have alterations in mRNA translation. De novo synthesis and half-life of cytosolic/nuclear proteins HFH4 and HNF3-alpha, membrane associated CYP2F2 and secreted proteins CC26 and trypC, will be evaluated by metabolic labeling of cultured microdissected airways from WT and CCSP [-/-] mice.
Aim 3 seeks to determine whether changes in oxidant sensitivity of CCSP [-/-] mice are due to CCSP deficiency, or to ultrastructural changes to clara cells with associated changes in secretion. To address these issues, CCSP [-/-] mice will be partially complimented either by in vivo retroviral gene transfer, or by use of a mouse surfactant protein C promoter-CCSP transgene. Retroviral CCSP gene transfer will allow assessment of trans-complementation of CCSP non-expressing airway regions by secretions from CCSP expressing airway regions. In contrast, SPC-CCSP transgene complementation will restore CCSP to the centri-acinar region without rectifying the clara cell secretory defect.
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